This project investigates the roles of viral DNA replication proteins in the interaction of herpes simplex virus (HSV) with the mammalian nervous system, especially virus latency. Latency is a fascinating biological property of the virus and, because latent virus is the source of recurrent disease, is an important clinical feature. Information about HSV latency is highly relevant to antiviral drugs, which target replication proteins, and vaccines, and might eventually permit the design of agents to cure herpesvirus infections. The proposed research will (aim 1) quantify configurations of HSV DNA during latency by pulsed-field gel electrophoresis coupled with polymerase chain reaction (PCR) and by ligation-mediated PCR. These highly sensitive methods will be applied to several problems including the configuration of wild type and mutant HSV DNA in ganglia. The second aim is to use these methods to follow the configurations of HSV DNA during the establishment of latency, especially by thymidine kinase (tk) mutants. Quantitative RNA PCR and cDNA cloning will be used to quantify and characterize HSV gene expression during the establishment of latency. The stage in the infectious cycle at which HSV replication is blocked during reaction from latency when TK or ribonucleotide reductase (RR) is absent or inhibited will be determined (aim 3). Ganglia infected with tk or rr null mutants or by wild type virus in the presence of TK-inhibitors will be examined for viral gene expression using in situ hybridization and PCR. Functions of HSV TK and non-essential functions of HSV DNA polymerase (Pol) that are important for pathogenesis in the peripheral and central nervous systems will be ascertained (aim 4). Recombinant viruses, in which the HSV tk gene is inactivated by insertion of a human tk or deoxycytidine kinase gene, and certain tk mutant viruses will be tested for ganglionic replication and reactivation and for neurovirulence. HSV pol mutants will be tested to determine if certain pol functions are particularly important for replication and pathogenesis in the brain. The proposed experiments should shed light on issues of virus and nervous system biology, regulation of gene expression, antiviral drug resistance, and the use of HSV as a vector or as therapy.